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As the Pope's personal chapel, Mass is frequently held inside, as are the Papal Conclaves, which occur after the Pope's death. The cardinals are locked inside the chapel and are not permitted to leave until a new Pope is elected.[1]

After an arduous fight with Rodrigo, in which two Pieces of Eden were used, Ezio was tricked and stabbed by Rodrigo, who had used the Papal Staff's power to immobilize the Assassin, before Ezio promptly fell unconscious.

Upon waking, Ezio pressed two hidden buttons at the back of the chapel, which opened the way into the Vault, a structure built by members of the First Civilization, whose entrance lay in a pit-like enclosure. It was at this entrance that Ezio would personally confront Rodrigo in a final, victorious fight.[1] Soon after the Assassin left the Vault, Mario Auditore appeared and beckoned him to leave the Capella Sistina, to which Ezio complied.[2]